Enzymatic activity of glycosyltransferase GLT8D1 promotes human glioblastoma cell migration

Ilina, Elena I.; Cialini, Camille; Gerloff, Dietlind L.; Garcia-Escudero, Maitane Duarte; Janty, Céline; Thézénas, Marie‐Laëtitia; Lesur, Antoine; Puard, Vincent; Bernardin, François; Moter, Alina; Schuster, Anne; Dieterle, Monika; Golebiewska, Anna; Gérardy, Jean-Jacques; Dittmar, Gunnar; Niclou, Simone P.; Müller, Tanja; Mittelbronn, Michel

doi:10.1016/j.isci.2022.103842

Cited by 7 publications

(33 citation statements)

References 51 publications

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“…Outcomes of major steps 1–3 form the basis for producing rationally designed active site mutants in the laboratory (major steps 4 and 5) whose intended (negative) impact on catalysis can be verified as it is described in major step 6. As reported in Ilina et al., 1 the resulting mutants ultimately served as tools to demonstrate a link between catalytic glycosyltransferase activity of GLT8D1 and cell migratory properties in glioblastoma (by procedures not included in this protocol).…”

Section: Before You Beginmentioning

confidence: 95%

“… Alternatives: Bioinformatics web resources change every now and then in appearance, in the tools or in the derived data that are offered. For example, in OrthoMCL release 5 that was used in our GLT8D1 study 1 we downloaded two OGs from the site so as to include the close paralog GLT8D2 [OG5_136216 and OG5_135167; last accessed November 2021] and aligned the sequences with a local installation of ClustalX 7 (step 3a). Using the current OrthoMCL website and data instead (release 6), an initial automated MSA can be generated directly using ClustalΩ 21 with selected sequences.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…

Figure 2 Phylogenetic consistency checking (step 10) (A) Example of a very well balanced tree that is consistent with species evolution; it was derived from the MSA we used to design GLT8D1 mutants. 1 No further corrections are required. Note: Minor topological differences, like those between the GLT8D1 and GLT8D2 mammalian groups, are common and tolerated because the amount of sequence variation is insufficient to expect stable, accurate positions in these subtrees).…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“… Structural/domain database PDB_mmCIF70_xxx Yes MSA generation iterations 0 MSA generation method HHblits=>UniRef30 Note: irrelevant if MSA generation iterations set to 0 as suggested above Yes E-value cut-off for MSA generation 1E-10 Note: irrelevant if MSA generation iterations set to 0 as suggested above Min seq identity of MSA hits with query 10% Min coverage of MSA hits 60% Secondary structure scoring during_alignment Yes Alignment mode:Realign with MAC local:norealign Yes MAC realignment threshold 0.3 Note: irrelevant parameter if previous parameter is set as suggested Yes Note: Parameter settings are shown as we used them to analyze GLT8D1 from the MSA shown in Ilina et al. 1 We prefer to set HHpred parameters slightly more conservatively than it is proposed by default but for most analyses the impact of this on the results will be minimal, i.e., different parameter settings may work equally well (or better) for your target. All parameters that are potentially relevant for this step are listed.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…The procedures detailed in this protocol should be adaptable to any other metal-dependent family within the diverse GT-A superfamily of glycosyltransferases and facilitate reproducing the mutant production and validation for GLT8D1 as reported. 1 Moreover, its strategic framework and various key resources are transferable to unrelated cases in principle, and its step-by-step sequence potentially serve as guidance for developing similar protocols for other enzymes with similar metal-dependence. Loss-of-function mutants are informative and affordable laboratory tools in practice, a.o.…”

Section: Before You Beginmentioning

confidence: 99%

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Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

et al. 2023

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Section: Before You Beginmentioning

confidence: 95%

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

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Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

et al. 2023

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Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase

Vicente,

Guerreiro,

Felgueiras

et al. 2023

Sci Rep

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Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn2+ and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes.

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Global organelle profiling reveals subcellular localization and remodeling at proteome scale

Hein,

Peng,

Todorova

et al. 2023

Preprint

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Defining the subcellular distribution of all human proteins and its remodeling across cellular states remains a central goal in cell biology. Here, we present a high-resolution strategy to map subcellular organization using organelle immuno-capture coupled to mass spectrometry. We apply this proteomics workflow to a cell-wide collection of membranous and membrane-less compartments. A graph-based representation of our data reveals the subcellular localization of over 7,600 proteins, defines spatial protein networks, and uncovers interconnections between cellular compartments. We demonstrate that our approach can be deployed to comprehensively profile proteome remodeling during cellular perturbation. By characterizing the cellular landscape following hCoV-OC43 viral infection, we discover that many proteins are regulated by changes in their spatial distribution rather than by changes in their total abundance. Our results establish that proteome-wide analysis of subcellular remodeling provides essential insights for the elucidation of cellular responses. Our dataset can be explored atorganelles.czbiohub.org.

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Enzymatic activity of glycosyltransferase GLT8D1 promotes human glioblastoma cell migration

Cited by 7 publications

References 51 publications

Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

Prediction and verification of glycosyltransferase activity by bioinformatics analysis and protein engineering

Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase

Global organelle profiling reveals subcellular localization and remodeling at proteome scale

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